WO2004010933A2 - Compositions cellulaires produisant de l'insuline et procedes associes - Google Patents

Compositions cellulaires produisant de l'insuline et procedes associes Download PDF

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WO2004010933A2
WO2004010933A2 PCT/US2003/023346 US0323346W WO2004010933A2 WO 2004010933 A2 WO2004010933 A2 WO 2004010933A2 US 0323346 W US0323346 W US 0323346W WO 2004010933 A2 WO2004010933 A2 WO 2004010933A2
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insulin
cell
cells
cell composition
producing
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PCT/US2003/023346
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WO2004010933A3 (fr
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Seung Kim
Ingrid Rulifson
Yuichi Hori
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The Board Of Trustees Of The Leland Stanford Junoir University
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Priority to AU2003252164A priority Critical patent/AU2003252164A1/en
Publication of WO2004010933A2 publication Critical patent/WO2004010933A2/fr
Publication of WO2004010933A3 publication Critical patent/WO2004010933A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/22—Coculture with; Conditioned medium produced by pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/32—Polylysine, polyornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • Diabetes mellitus is a major cause of morbidity and mortality worldwide, and incidence rates of type I and type II DM are increasing.
  • type I DM destruction of insulin- producing pancreatic islets leads to a prolonged illness often culminating in devastating multisystem organ failure and early mortality.
  • Clinical trials demonstrate that tight glucose regulation can prevent the development of diabetic complications, but attempts to achieve this regulation by exogenous insulin administration are only partially successful.
  • islet cell transplantation with improved systemic immunosuppression may provide a short-term durable remission in insulin requirements in type I diabetics (Shapiro et al, 2000, N Engl J Med. 343: 230-238 ; Ryan et al, 2001, Diabetes 50: 710-719).
  • An expandable source of tissues like human stem cells may provide the best promise for tissue replacement strategies for human diseases.
  • Stem cells including embryonic stem (ES) cells and various adult stem cells provide a promising potential means for cell-replacement therapy in human diseases.
  • Stem cells may provide serve as an inexhaustible source for the production of replacement islets for transplantation in diabetic humans.
  • ICCs stably-differentiated functional insulin-producing cell compositions
  • Methods to provide a renewable source of replacement islets from stem cells could transform therapeutics in DM.
  • the invention relates to methods for making insulin-producing cell compositions by a method that comprises culturing a suitable cell composition in the presence of a cell proliferation inhibitor. In certain embodiments, the invention relates to methods for making insulin-producing cell compositions by a method that comprises culturing a suitable cell composition in the presence of a phosphatidylinositol 3' -kinase (PI3K) pathway inhibitor.
  • the suitable cell composition is a stem-cell derived cell composition, such as a composition derived from a mammalian embryonic stem cell line or adult stem cell line.
  • the stem cell-derived cell composition comprises a detectable amount of a marker that is indicative of neural, hepatic or pancreatic cells.
  • the suitable cell composition is cultured in the presence of both a PI3K pathway inhibitor and a poly adenosine diphosphate ribosyl transferase (ADPRT) inhibitor, such as a nicotinamide agent or a benzamidine.
  • ADPRT poly adenosine diphosphate ribosyl transferase
  • a method of making an insulin-producing cell composition comprises culturing a cell composition comprising stem cells so as to produce a cell composition comprising cells expressing one or more markers that are indicative of neural cells, hepatic cells or pancreatic cells, and then further culturing the cell composition in the presence of a cell proliferation inhibitor to obtain an insulin-producing cell composition.
  • the stem cells are from a stem cell line.
  • the one or more markers are selected from the group consisting of: nestin, neurogenin, islet- 1, glucagon and insulin.
  • the cell line is initially cultured in the presence of leukemia inhibitory factor, or a functional variant thereof.
  • the cell composition is cultured in the presence of insulin, transferrin, selenium and fibronectin, or functional analogs thereof, followed by culturing in the presence of basic fibroblast growth factor, or a functional analog thereof.
  • the cell composition comprising cells expressing one or more markers that are indicative of neural cells, hepatic cells or pancreatic cells, is cultured in the presence of both a cell proliferation inhibitor and a poly adenosine diphosphate ribosyl transferase (ADPRT) inhibitor, such as a nicotinamide agent or a benzamidine.
  • ADPRT poly adenosine diphosphate ribosyl transferase
  • a method of making an insulin-producing cell composition comprises culturing a cell composition comprising stem cells so as to produce a cell composition comprising cells expressing one or more markers that are indicative of neural cells, hepatic cells or pancreatic cells, and then further culturing the cell composition in the presence of a PI3K pathway inhibitor to obtain an insulin-producing cell composition, h certain embodiments, the stem cells are from a stem cell line.
  • the one or more markers are selected from the group consisting of: nestin, neurogenin, islet- 1, glucagon and insulin.
  • the cell line is initially cultured in the presence of leukemia inhibitory factor, or a functional variant thereof.
  • the cell composition is cultured in the presence of insulin, transferrin, selenium and fibronectin, or functional analogs thereof, followed by culturing in the presence of basic fibroblast growth factor, or a functional analog thereof.
  • the PI3K pathway inhibitor is a PI3K inhibitor, such as an agent that inhibits PI3K activity or an agent that decreases the level of PI3K protein subunit(s) in the cell.
  • an insulin-producing cell composition comprises at least about 1000 nano grams of insulin per milligram of total protein, and preferably at least about 5000 or 10000 nano grams of insulin per milligram of total protein.
  • an insulin-producing cell composition of the invention is such that at least about 80%, 90%, 95% or 99% of the cells of the insulin-producing cell composition produce insulin.
  • the invention relates to therapeutic cell compositions comprising an insulin-producing cell composition disclosed herein and a therapeutically acceptable excipient.
  • the insulin-producing cell compositions may also, in certain embodiments, b e used for therapeutic purposes without an excipient.
  • C ertain aspects o f the invention also relate to methods of making a therapeutic cell compositions, comprising mixing an insulin-producing cell composition with a therapeutically acceptable excipient.
  • the invention provides for the use of an msulin-producing cell composition d isclosed h erein for t he m anufacture o f a m edicament for t he amelioration o f a condition related to insufficient pancreatic function, such as a form of diabetes.
  • the insulin-producing cell composition is mixed or otherwise combined with a therapeutically acceptable excipient.
  • the insulin-producing cell compositions may also, in certain embodiments, be used for therapeutic purposes without an excipient.
  • An insulin-producing cell composition for in the manufacture of a medicament may be an insulin-producing cell composition generated according to any of the methods disclosed herein.
  • the insulin-producing cell composition comprises at least about 1000 nanograms of insulin per milligram of total protein, and preferably at least about 5000 or 10000 nanograms of insulin per milligram of total protein.
  • the insulin-producing cell composition is such that at least about 80%, 90%, 95% or 99% of the cells of the insulin-producing cell composition produce insulin.
  • the invention relates to methods of ameliorating, in a subject, a condition related to insufficient pancreatic function, such as a form of diabetes.
  • a method comprises administering to a subject an effective amount of an insulin producing cell composition disclosed herein or made according to a method disclosed herein.
  • the administration of an insulin-producing cell composition causes an increase in blood insulin levels in the subject. In further preferred embodiments, the administration of an insulin-producing cell composition improves blood glucose homeostasis. In yet additional preferred embodiments, the insulin producing cell composition c auses a neoplasm in fewer than 30% or 1 5% o f subjects and, in a p articularly preferred embodiment, fewer than 5% or fewer than 1% of subjects. In certain embodiments, the invention relates to methods for testing the developmental potential of a cell of interest.
  • An embodiment of such a method comprises subjecting a stem cell line to an in vitro differentiation process so as to cause a cell of the stem cell-derived cell composition to give rise to a cell that produces a pancreatic hormone, mixing a cell of interest with the stem cell-derived cell composition at a point in the differentiation process and determining the identity of cells derived from the cell of interest, thereby testing the developmental potential of the cell of interest.
  • the cell of interest is obtained from a pancreatic tissue, and in preferred embodiments the method is operated repeatedly (in series, in parallel, orb oth) t o a ssess the d evelopmental p otential o f a v ariety o f cells o btained from a pancreatic tissue, h certain embodiments, the in vitro differentiation process comprises contacting the stem cell-derived cell composition with a cell proliferation inhibitor. In a preferred embodiment, the in vitro differentiation process comprises contacting the stem cell- derived cell composition with a PI3K pathway inhibitor.
  • methods for testing developmental potential comprise forming a mixed cell composition comprising the cell of interest and a cell composition suitable for culturing in the presence of a PI3K inhibitor to create a mixed cell composition, culturing the mixed stem cell-derived cell composition in the presence of a PI3K pathway inhibitor, thereby obtaining a mixed insulin-producing cell composition; and determining the identity of cells derived from the cell of interest, thereby testing the developmental potential of the cell of interest.
  • the cell of interest is mixed with the starting cell culture (e.g. cells of a stem cell line) or at another stage of the differentiation process .
  • the invention relates to methods for identifying a candidate gene related to pancreatic development.
  • An embodiment of such a method comprises measuring a first level of a gene product in a stem cell-derived cell composition, culturing the stem-cell derived cell composition in the presence of a PI3K pathway inhibitor, thereby obtaining an insulin-producing cell composition, and measuring a second level of the gene product in the msulin-producing cell composition, wherein a gene that encodes the gene product that has a significantly different first and second levels is a candidate gene related to pancreatic development.
  • Gene products include transcripts, proteins, post-translational modifications etc., and may be measured, for example, by nucleic acid or protein array systems (e.g. microarrays) or other methods such as two-dimensional gel electrophoresis, mass spectroscopy, etc.
  • insulin-producing cell compositions may be contacted with a test compound to screen for compounds that have desired effects, such as proliferative or insulin-production promoting effects, on insulin-producing cells. It is further contemplated that insulin-producing cells may be genetically modified. For example, insulin-producing cells may be genetically modified to become hyperproliferative, and such hyperproliferative cells may be used to test for anti-neoplastic agents.
  • FIG. 1 Similarities of gene expression and mitotic status in pancreatic ⁇ cells and insulin- producing cells comprising stage 5NL ICCs. Images were obtained by confocal microscopy from at least 3 samples for each immunohistochemical probe. Simultaneous immunofiuorescent detection of insulin and (A-C) glucagon, (D-F) ⁇ -fetoprotein ( ⁇ -FP), (G-I) MAP2, or (J-L) ⁇ - tubulinlll ( ⁇ tublll). Insulin staining appears as a pale gray in each box, while glucagon, ⁇ -FP, MAP2, or ⁇ tublll appear as darker gray.
  • Panel R inset shows a merge (lighter gray) of simultaneous immunohistochemical detection of insulin (pale gray) and C-peptide expression (dark gray) in these engrafted ICCs; most of the signal indicates overlap in insulin and C-peptide expression.
  • Stage 5NL ICCs express several characteristic pancreatic ⁇ cell markers.
  • Immunofiuorescent staining of adult pancreatic islets and stage 5NL ICCs for insulin and C- peptide (A and B) (showing substantial overlap in expression of these markers), insulin and proinsulin (C and D) (showing interspersed insulin positive cells and proinsulin positive cells in the islet, with a somewhat greater extent of overlap in the 5 NL ICCs), Pdxl (E and F) (dark gray, abundant in both fields), Glut-2 (G and H) (dark gray, abundant in each field), and glucokinase (I and J) (dark gray, abundant in both fields).
  • pancreatic islet preparations K, RT-PCR analysis of ICC gene expression during stages 1-4, and in hand-isolated 5NB and 5NL ICCs, and wildtype pancreatic islets.
  • Islet- 1 and neurogenin-3 are transcription factors required for islet formation in mice (25, 26) and Nestin is an intermediary filament protein (2).
  • Nestin is an intermediary filament protein (2).
  • stage 5 ICCs applicants detected carboxypeptidase A, but not pancreatic amylase, both products of pancreatic exocrine (acinar) cells.
  • Amylase transcripts are routinely detected by RT-PCR in pancreatic islet preparations because of adherent acinar cells (13). Other gene products are described in the examples, below.
  • Transplanted stage 5NL ICCs function in vivo to rescue and ameliorate diabetic phenotypes.
  • an element means one element or more than one element.
  • adult stem cell is used herein to refer to a stem cell obtained from any non- embryonic tissue.
  • stem cells derived from fetal tissue and amniotic or placental tissue are included in the term adult stem cell.
  • Cells of these types tend to have properties more similar to cells derived from adult animals than to cells derived from embryonic tissue, and accordingly, for the purposes of this application stem cells may be sorted into two categories: “embryonic” and “adult” (or, equivalently, "non-embryonic”).
  • a "patient” or “subject” to be treated by the method of the invention can mean either a human or non-human animal, preferably a mammal.
  • a “cell composition” is any composition of matter g enerated by human manipulation that comprises viable cells as a substantial component.
  • a cell composition may comprise more than one type of viable cell.
  • An “enriched cell composition” is a cell composition comprising a substantially greater purity (i.e. at least twice as pure) of a recognizable cell type than is found in any natural tissue.
  • a “pure cell composition” is a cell composition that comprises at least about 75%, and optionally at least about 85%, 90% or 95% of a recognizable cell type.
  • a recognizable cell type is generally one that has a reasonably uniform morphology, a characteristic set of two or more molecular markers and a functional characteristic. It is understood that there is likely to be some variation in certain characteristics even within a recognizable cell type.
  • a cell composition may comprise, in addition to cells, essentially any component(s) that are compatible with the intended use for the cell composition.
  • a cell composition may include media, growth factors, pharmaceutically acceptable excipients, preservatives, a solid or semi-solid substrate, a porous matrix or scaffold, nonviable cells or a therapeutic agent.
  • a "cyclic AMP stimulating agent” or “cAMP stimulating agent” is any agent that causes an increase in cAMP mediated cell signaling.
  • exemplary cyclic AMP stimulating agents include forskolin and membrane diffusible cAMP analogues and phosphodiesterase inhibitors including 3-isobutly-l -methyl xanthine (IBMX).
  • a later cell is "derived" from an earlier cell if the later cell is descended from the earlier cell through one or more cell divisions. Where a cell culture is initiated with one or more initial cells, it may be inferred that cells growing up in the culture, even after one or more changes in culture condition, are derived from the initial cells. A later cell may still be considered derived from an earlier cell even if there has been an intervening genetic manipulation.
  • culturing includes exposing cells to any condition. While “culturing” cells is often intended to promote growth of one or more cells, “culturing” cells need not promote or result in any cell growth, and the condition may even be lethal to a substantial portion of the cells.
  • insulinogenic agent is an agent that promotes proliferation, maturation, survival or differentiation of insulin producing cells, or that promotes insulin production, maturation, storage or secretion.
  • exemplary insulinogenic agents include: IGF-1, HGF, a cyclic AMP stimulating agent, exendin, GLP1, PPAR ⁇ ligand, sonic hedgehog, PACAP and growth hormone.
  • LY294002 refers to the phosphatidylinositol 3-kinase inhibitor, 2-(4-M ⁇ holinyl)-8-phenyl-4 H-l-benzopyran-4-one; as described by Vlahos, et al. (1994) J. Biol., Chem., 269(7) 5241-5248, and is available from Calbiochem Corp., La Jolla Calif.
  • an insulin marker refers to a detectable aspect of a cell.
  • an insulin marker may include an insulin transcript or an insulin polypeptide, such as proinsulin, the alpha chain, the beta chain or the C peptide.
  • a cell is "positive” for a marker if that marker is convincingly detected in the cell.
  • nicotinamide agent includes nicotinamide and analogs thereof that are biocompatible.
  • a nicotinamide agent has ADPRT inhibitory activity.
  • percent identical refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Percent identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison.
  • a s a p ercentage o f i dentity refers t o a function o f t he n umb er o f i dentical a mino acids or nucleic acids at positions shared by the compared sequences.
  • Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ.
  • FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin,
  • ENTREZ is available through the
  • the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
  • MPSRCH uses a Smith- Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.
  • PI3K refers to a phosphatidylinositol (PI) 3'-kinase, a family of proteins that phosphorylate the inositol ring of PI in the D-3 position.
  • the canonical mammalian PI3K is a heterodimeric complex that contains p85 and a 110-Kd protein (pi 10) (Carpenter et al. (1990) J.
  • the purified p85 subunit has a regulatory role while the 110-Kd subunit harbors the catalytic activity.
  • the "PI3K pathway” refers to the collection of molecular actors that are influenced by the primary effects of PI3K activity, as well as the collection of molecular actors that have primary effects on PI3K activity. It is understood that a cellular signaling event, such as an increase in the activation of a PI3K, will generally produce effects that occur independent of any changes in gene expression, referred to herein as "primary” effects, such as certain changes in protein-protein interactions, certain changes in protein activities and certain changes in post- translational modifications.
  • the PI3K pathway is not intended to encompass the vast array of actors that become involved only as a result of changes in gene expression.
  • a "PI3K pathway inhibitor” is any substance or mixture of substances that decreases a cellular activity that is increased in response to PI3K activity.
  • a PI3K pathway inhibitor may be a " PI3K i nhibitor", m eaning t hat t he i nhibitor a cts b y d ecreasing P I3K a ctivity.
  • a P I3K inhibitor may, for example, inhibit an activity of a PI3K enzyme or decrease the level of protein of one or more PI3K subunits..
  • a PI3K pathway inhibitor may act by affecting a part of the PI3K pathway.
  • Exemplary PI3K pathway inhibitors include wortmannin, LY294002, a PI3K-targeted RNAi, etc.
  • a "poly-adenosine diphosphate ribosyl transferase inhibitor” or "ADPRT inl ibitor” includes any compound or treatment that inhibits the ADPRT enzyme.
  • Exemplary ADPRT inhibitors include nicotinamide and N-substituted benzamidines.
  • stem cell refers to an undifferentiated cell which is capable of proliferation and giving rise t o at 1 east o ne m ore d ifferentiated c ell t ype.
  • Totipotent s tem cells are stem cells that are capable of giving rise to any cell type of the organism from which the stem cells were obtained.
  • Pluripotent stem cells are stem cells that are capable of giving rise to cells of the three major embryonic lineages, the endoderm, mesoderm and ectoderm.
  • Multipotent stem cells are stem cells that are capable of giving rise to more than one type of more differentiated cell.
  • stem cell is also intended to include cells of varying developemental potential that may be obtained by somatic cell nuclear transfer or by causing a differentiated cell to undergo de-differentiation.
  • a stem cell is named by the tissue from which it was obtained.
  • an "embryonic stem cell” is a stem cell obtained from an embryo and a “pancreatic stem cell” is a stem cell obtained from a pancreas, understanding that many different stem cell types may usually be obtained from a single tissue source.
  • a “stem cell line” is an enriched or pure cell composition comprising a recognizably distinct stem cell type that, when cultured in appropriate conditions, self-propagates.
  • stem cell line is explicitly intended to include cell compositions that have not been genetically manipulated or extensively passaged, just so long as there has been enrichment for stem cells.
  • a “stem cell line-derived cell composition” or (in shorter but interchangeable form) a “stem cell-derived cell composition” is a cell composition that is obtained by culturing a stem cell line to obtain the desired cell composition.
  • a stem cell line may be cultured through a series of different culture conditions or selections (such as fluorescence activated cell sorting or other methods for separating different cell types) to obtain the stem cell-derived cell composition.
  • the invention relates to methods of generating insulin-producing cell compositions by exposing a suitable cell composition to an inhibitor of cell proliferation.
  • a suitable cell composition is exposed to an inhibitor of the PI3K pathway.
  • a suitable cell composition is one that, when exposed to an effective amount of an inhibitor of cell proliferation in an appropriate medium develops increased insulin production.
  • the suitable cell composition is a stem cell-derived cell composition.
  • a stem cell-derived cell composition for use in the methods disclosed herein may be essentially any composition of cells that results from culturing cells of a stem cell line.
  • Suitable stem cell lines include embryonic stem cell lines and adult stem cell lines, whether totipotent, pluripotent, multipotent or of lesser developmental capacity.
  • Stem cell lines are preferably derived from mammals, such as rodents (e.g. mouse or rat), primates (e.g.
  • mouse embryonic stem cells include: the JM1 ES cell line described in M. Qiu et al., Genes Dev 9, 2523 (1995), and the ROSA line described in G Friedrich, P. Soriano, Genes Dev 5, 1513 (1991), and mouse ES cells described in US Patent No. 6,190,910. Many other mouse ES lines are available from Jackson Laboratories (Bar Harbor, Maine).
  • human embryonic stem cells include those available through the following suppliers: Arcos Bioscience, Inc., Foster City, California, CyThera, Inc., San Diego, California, BresaGen, Inc., Athens, Georgia, ES Cell International, Melbourne, Australia, Geron Corporation, Menlo Park, California, G ⁇ teborg University, G ⁇ teborg, Sweden, Karolinska Institute, Sweden, Maria Biotech Co. Ltd.
  • embryonic stem cells are described in the following U.S. patents and published patent applications: 6,245,566; 6,200,806; 6,090,622; 6,331,406; 6,090,622; 5,843,780; 20020045259; 20020068045.
  • the human ES cells are selected from the list of approved cell lines provided by the National Institutes of Health and accessible at http://escr.nih.gov.
  • Examples of human adult stem cells include those described in the following U.S. patents and patent applications: 5,486,359; 5,766,948; 5,789,246; 5,914,108; 5,928,947; 5,958,767; 5,968,829; 6,129,911; 6,184,035; 6,242,252; 6,265,175; 6,387,367; 20020016002; 20020076400; 20020098584; and, for example, in the PCT application WO 0111011.
  • a stem cell line is selected from the group consisting of: the WA09 line obtained from Dr. J. Thomson (Univ. of Wisconsin) and the UC01 and UC06 lines, both on the current NTH registry.
  • an adult stem cell line is a neurosphere-type neural stem cell line or a multipotential adult stem cell as described by Verfaillie et al. in WO 0111011.
  • a suitable cell composition may be derived from a cell fusion or dedifferentiation process, such as described in the following US patent application: 20020090722, and in the following PCT applications: WO200238741, WO0151611, WO9963061, WO9607732.
  • a stem cell line may include cells cultured directly from a tissue sample in such a way as to enrich for one or more types of stem cells.
  • a passaged stem cell line is one that has been propagated through at least two media changes or growth substrate changes since being obtained from a tissue sample.
  • a stem cell line should be compliant with good tissue practice guidelines set for the by the U.S. Food and Drag Administration (FDA) or equivalent regulatory agency in another country. Methods to develop such a cell line may include donor testing, and avoidance of exposure to non-human cells and products during derivation of the stem cell lines.
  • FDA U.S. Food and Drag Administration
  • the stem cell line can be prepared and used without the use of a feeder layer or any type of virus or viral vector.
  • the cells of the insulin-producing cell composition have a wild-type genotype, meaning that the genotype of the cells is a genotype that may be found in a subject organism naturally.
  • the genotype of the cells is a genotype that may be found in a subject organism naturally.
  • cells having chromosomal rearragements as a result of culture treatments are not cells having a wild-type genotype.
  • cells that have been transfected with an integrating nucleic acid construct will not (except in cases of perfect excision) have a wild-type genotype.
  • the term "genotype" does not refer to peripheral modifications to the genomic nucleic acids, such as methylation, and therefore, cells having a naturally occurring genetic makeup may have unnatural phenotypes as an effect of changes in methylation or other modifications.
  • the suitable cell composition such as a stem cell-derived cell composition
  • is positive for markers that are traditionally associated with neural cell types referred to herein as "markers that are indicative of neural cell types"
  • markers that are indicative of neural cell types such as: nestin, neurogenin, MAP2 and beta-tubulin III.
  • at least about 5%, at least about 20%>, at least about 50%, at least about 75%, or at least about 90% of the cells of the stem cell-derived cell composition are positive for a marker that is indicative of a neural cell type.
  • the stem cell-derived cell composition is positive for markers that are traditionally associated with pancreatic cell types (referred to herein as "markers that are indicative of pancreatic cell types”) such as: insulin (including proinsulin, alpha chain, beta chain or C peptide), glucagon, GLUT2, islet amyloid polypeptide (IAPP), glucokinase, HNF3 ⁇ , HNFl ⁇ , HNF4 ⁇ , HNF3 ⁇ , or PDX1 (also known as IPF1, STF1 and IDX1).
  • markers that are traditionally associated with pancreatic cell types such as: insulin (including proinsulin, alpha chain, beta chain or C peptide), glucagon, GLUT2, islet amyloid polypeptide (IAPP), glucokinase, HNF3 ⁇ , HNFl ⁇ , HNF4 ⁇ , HNF3 ⁇ , or PDX1 (also known as IPF1, STF1 and IDX1).
  • the stem cell-derived cell composition is positive for markers that are traditionally associated with hepatic cell types or "markers that are indicative of hepatic cell types", such as:, GLUT2, HNF3 ⁇ , HNFl ⁇ , HNF4 ⁇ , HNF3 ⁇ , alpha-fetoprotein and glucokinase.
  • markers that are traditionally associated with hepatic cell types or "markers that are indicative of hepatic cell types"
  • markers are traditionally associated with hepatic cell types or "markers that are indicative of hepatic cell types”
  • markers are associated with both pancreatic and hepatic lineages.
  • insulin-producing cell compositions may be derived from hematopoietic or mesenchymal stem cells, such as stem cell populations dervied from adult human bone marrow.
  • HSCs marrow-derived hematopoietic
  • MSCs mesenchymal stem cells
  • Purified HSCs not only give rise to all cells in blood, but can also develop into cells normally derived from endoderm, like hepatocytes (Krause et al., 2001, Cell 105: 369-77; Lagasse et al., 2000 Nat Med 6: 1229-34).
  • endoderm like hepatocytes
  • MSCs appear to be similarly multipotent, producing progeny that can, for example, express neural cell markers (Pittenger et al., 1999 Science 284: 143-7; Zhao et al., 2002 Exp Neurol 174: 11-20).
  • insulin-producing cell compositions are derived from an autologous source or an HLA-type matched source.
  • HSCs may be obtained from the bone marrow of a subject in need of msulin-producing cell compositions and cultured by a method described herein to generate an autologous insulin-producing cell compositions.
  • Other sources of stem cells are easily obtained from a subject, such as stem cells from muscle tissue, stem cells from skin (dermis or epidermis) and stem cells from fat.
  • Insulin producing cell compositions may also be derived from banked stem cell sources, such as banked amniotic epithelial stem cells or banked umbilical cord blood cells.
  • Certain embodiments of the methods disclosed herein are advantageous in part because they permit the generation of insulin-producing cell compositions from starting materials, such as many of the passaged stem cell lines identified herein, that are available, as a practical matter, in sufficient quantities for formation o f a therapeutically e ffective insulin-producing implant.
  • starting materials such as many of the passaged stem cell lines identified herein
  • fetal pancreatic tissue and particularly human fetal pancreatic tissue, is only available in small quantities, making it difficult or impossible to assemble sufficient material to form a therapeutically effective implant.
  • a stem cell-derived cell composition suitable for treatment with a PI3K pathway inhibitor may be generated according to the following steps.
  • cells of the stem cell line are cultured in the presence of leukemia inhibitory factor (LIF), or a functional analog thereof.
  • LIF leukemia inhibitory factor
  • the cells are cultured on a feeder layer, and exemplary feeder layers include irradiated fibroblasts, preferably species-matched (e.g. mouse fibroblasts for mouse stem cells, human fibroblasts for human stem cells) and Matrigel (BD Biosciences, Lexington, Connecticut).
  • LLF leukemia inhibitory factor
  • the cells are cultured in the absence of the LLF (or LIF functional analog).
  • the cells are also cultured in the absence of the feeder layer on an adhesive or non- adhesive surface.
  • the cells are cultured in the presence of ITSFn (a mixture of insulin, transferrin, selenium and fibronectin, or functional analogs thereof).
  • ITSFn a mixture of insulin, transferrin, selenium and fibronectin, or functional analogs thereof.
  • An example of an ITSFn medium is described in Lee et al. Nature Biotechnol. 18: 675 (2000).
  • the resulting cells are cultured in the presence of basic fibroblast growth factor and neurotrophic factors (e.g. B27 supplement), or functional analogs thereof, thereby generating a stem cell-derived cell composition for use in making an insulin-producing cell composition.
  • basic fibroblast growth factor and neurotrophic factors e.g. B27 supplement
  • a suitable stem cell-derived cell composition or other suitable cell composition may be cultured in the presence of an amount of a cell proliferation inhibitor sufficient to decrease the rate of cell proliferation.
  • a PI3K pathway inhibitor may be used to achieve an inhibition of cell proliferation.
  • a PI3K inhibitor may also be selected for effects that are independent of effects on cell proliferation.
  • a cell proliferation inhibitor may be selected as agent that increases the expression and/or activity of one or more cell cycle inhibitors, such as cyclin-dependent kinases (cdks), e.g., pl5, pl7, pl8, p21, p27, or p57.
  • cdks cyclin-dependent kinases
  • GDFll is a preferred cell proliferation inhibitor.
  • the cell composition is also contacted with an inhibitor of ADPRT, such as a nicotinamide agent or a benzamidine agent.
  • the cell composition is contacted with the PI3K pathway inhibitor in the absence of B27 supplement, optionally in the absence of neurotrophic factors.
  • a variety of PI3K pathway inhibitors may be employed in the methods disclosed herein, including direct inhibitors that act directly on a PI3K subunit (or decrease the amount thereof) and pathway inhibitors that affect one or more other molecular actors in the PI3K pathway.
  • PI3K pathway inhibitors may have both direct and indirect actions.
  • Phosphatidylinositol 3-kinase has been found to phosphorylate the 3-position of the inositol ring of phosphatidylinositol (PI) to form phosphatidylinositol 3-phosphate (PI-3P) (Whitman et al.(1988) Nature, 322: 664-646).
  • this enzyme also can phosphorylate phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate to produce phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (PIP3), respectively (Auger et al. (1989) Cell, 57: 167-175).
  • Direct PI3K inhibitors include materials that reduce or eliminate either or both of these activities of P3 K. Such inhibitors include direct PI3K inhibitors such as Ly294002 (Calbiochem Corp., La Jolla, Calif.) and wortmannin (Sigma Chemical Co., St. Louis Mo.
  • Ly294002 The chemical properties of Ly294002 are described in detail in J. Biol, Chem., (1994) 269: 5241-5248. Ly294002 is stable in tissue culture medium and is membrane permeable. Wortmannin tends to degrade in cell cultures with a half-life of about 4-5 hours.
  • inhibitors of PI3K include viridin, viridiol, demethoxyviridin, and demethoxyviridiol (see, U.S. Pat. No. 5,276,167). Once viridin, viridiol, demethoxyviridin, and demethoxyviridiol, or other PI3K inhibitors are isolated and purified, analogs of each may be prepared via well known methods to provide generally known compounds such as those illustrated by formula I of U.S. Pat. No. 5,276,167 (see, also, Grove et al. (1965) J. Chem. Soc, June: 3803-3811, Hanson et al. (1985) J. Chem. Soc. Perkin Trans. I: 1311-1314. Aldridge et al. (1975) J. Chem. Soc. Perkin Trans. I: 943-945 (1975), and Blight et al. J. Chem. Soc. Perkin Trans I: 1317-1322).
  • Suitable derivatives and analogues include, but are not limited to alpha/beta- viridin, 1- acetylviridin, 1-methylether of viridin, demethoxyviridin, demethoxyviridin mono-acetate, dehydroxyviridin, demethoxyviridin mono-methanesulfonate, diacetyldemethoxyviridol OAc, viridiol, 1-O-acetylviridiol, 1-O-methyl-methylether of viridiol, demethoxyviridiol, 1- acetyldemethoxyviridiol, 1-O-methylether dimethoxyviridiol (see, U.S. Pat. No. 5,276,167).
  • Other derivatives include, but are not limited to Wortmannin stereochemical alcohol and ester derivatives, such as 11 -substituted, 17-substituted and 11, 17 disubstituted derivatives of wortmannin (see, U.S. Pat. No. 5,480,906), and the like.
  • Direct PI3K inhibitors may also include nucleic acid or antibody reagents (or other targeted polypeptide or peptide analogs) that are directed against a PI3K subunit (e.g. p85 or pi 10) or a nucleic acid encoding the subunit.
  • Nucleic acid inhibitors may include RNAi oligonucleotides and antisense nucleic acids.
  • An antisense construct can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a PI3K pathway protein.
  • the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of a PI3K pathway gene.
  • Oligonucleotides that are complementary to the 5' end of the mRNA should work most efficiently at inhibiting translation.
  • sequences complementary to the 3' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well.
  • antisense nucleic acids should be at least six nucleotides in length, and are preferably less that about 100 and more preferably less than about 50, 25, 17 or 10 nucleotides in length.
  • RNAi Inhibitor RNA
  • siRNA may also be used to inhibit a component of the P I3K p athway, and in certain embodiments, RNAi approaches may be d esirable b ecause such approaches are thought to be temporary and to cause no permanent changes in the genome of the target cell.
  • RNAi may be achieved as described in any of the following patent applications: WO9932619, WO0044914, WO0129058, WO0175164, and WO0136646.
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209)
  • methylphosphonate olgonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
  • Antibody reagents may include single chain antibodies or other antibody forms that can be introduced into a cell, either as a protein or as a nucleic acid competent to express the protein, to act directly on PI3
  • Targets for indirect inhibitors include PI3K pathway proteins, such as insulin receptor,
  • IGF receptors protein kinase C (PKC), IRS proteins, protein kinase B (PKB or Akt), NFAT
  • mitogen- activated protein kinases MTOR
  • GSK3 calcineurin
  • Stem cell lines may be engineered to permit conditional expression of PI3K or actors in the pathway.
  • a cell line may be engineered with the relevant gene placed under control of an inducible promoter, such as a tet promoter.
  • a cell line may be engineered with the relevant gene placed between recombinase sites (such as lox sites for the Cre recombinase) that permit excision upon expression of Cre, and Cre expression may be controlled by an inducible promoter.
  • PI3K inhibitor may be assessed according to any of a variety of art-recognized assays, hi general, the assays for direct PI3K inhibitors involve comparing the activity of PI3K in the presence and absence of the putative inhibitor. Examples of such assays are described in detail in U.S. Pat. No. 5,480,906. PI3K activity may be measured as described by Matter et al. (1992) Biochem. Biophys. Res. Comm., 186: 624-631.
  • inhibitor candidates are initially dissolved, e.g., in DMSO and then diluted e.g., 10-fold with 50 mM of HEPES buffer, pH 7.5, containing 15 mM of MgC12 and 1 mM of EGTA. Ten microliters of this solution are incubated with purified PI3K and phosphatidylinositol in 50 mM of HEPES buffer, pH 7.5, containing 1 mM of EGTA.
  • Reactants are preincubated 10 minutes at ambient temperature and then the enzyme reaction is started upon addition of 32P-ATP (2 mCi/mL, 500 mu M of stock solution; 0.08 mCi/mL, 20 mu M of final concentration; Dupont New England Nuclear, Boston, Mass.). The reaction is allowed to proceed for 10 minutes at ambient temperature with frequent mixing, after which time the reaction is quenched by addition of 40 mu L of IN HC1. Lipids are extracted with addition of 80 ⁇ l L CHC13 :MeOH (1:1, v:v).
  • the samples are mixed and centrifuged, and the lower organic phase is applied to a silica gel TLC plate (EM Science, Gibbstown, N.J.), which is developed in CHC13 :MeOH:H2O:NH4OH (45:35:8.5:1.5, v:v). Plates are dried, and the kinase reaction visualized by autoradiography. The phosphatidylinositol 3-monophosphate region is scraped from the plate and quantitated using liquid scintillation specfroscopy with ReadyProtein (Beckman Instruments, Inc., Fullerton, Calif.) used as the scintillation cocktail. The level of inhibition for the putative inhibitor is determined as the percentage of 32P-counts per minute compared to controls.
  • the activity of a candidate PI3K inhibitor may also be assessed by testing its effect on expression of a PI3K-responsive gene in a cell, particularly where the promoter of the PI3K- responsive gene is linked to a reporter gene.
  • a functional analog is a structurally similar molecule having at least 10%, and preferably at least 50%>, of the activity of the inhibitor or reagent.
  • a functional analog may be simply a version using one or more modified amino acids but retaining the same sequence, or a functional analog may be a polypeptide having at least
  • a combinatorial chemical library is a collection of diverse chemical compounds. Such libraries may be generated by chemical synthesis or biological synthesis by combining a number of simpler chemical subunits. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of amino acids in as many ways as possible for a given polypeptide length. The functionality of a candidate functional analog may be evaluated by using a published assay for the activity of the agent to be replaced. Millions of chemical compounds can be synthesized through such combinatorial mixing of subunits.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88).
  • Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, Dec.
  • nucleic acid libraries see, e.g., Strategene, Corp.
  • peptide nucleic acid libraries see, e.g., U.S. Pat. No. 5,539,083
  • antibody libraries see, e.g., Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314
  • PCT/US96/10287 carbohydrate libraries
  • carbohydrate libraries see, e.g., Liang et al. (1996) Science, 274: 1520- 1522, and U.S. Pat. No. 5,593,853
  • small organic molecule libraries see, e.g., benzodiazepines, Baum (1993) C&EN, January 18, page 33, isoprenoids U.S.
  • an insulin-producing cell composition is exposed to an additional culture condition.
  • An additional culture condition may be simply the removal of the PI3K pathway inhibitor or cell proliferation inhibitor.
  • the removal of the inhibitor stimulates growth of cells in the insulin-producing cell composition and increased insulin production, h addition or independent of removal of the inhibitor, the insulin- producing cell composition may be treated with any of the various agents, and functional analogs t hereof, t hat a re k nown t o s timulate i nsulin p roduction or b eta-cell p roliferation.
  • such agents are only effective in increasing insulin production if used subsequent to or simultaneously with the PI3K pathway inhibitor of cell proliferation inhibitor.
  • agents include Igf-1 (e.g. at a concentration of lOng/ml), GLP-1, exendin-4, HGF, activin, sonic hedgehog (and other suitable hedgehog family members, as well as hedgehog agonists) and reagents that increase cAMP levels, such as membrane permeable forms of cAMP and forskolin.
  • Insulin-Producing Cell Compositions In certain embodiments, the invention relates to insulin-producing cell compositions produced according to any of the methods disclosed herein. Insulin-producing cell compositions may be in any form, including, preferably, insulin-producing cell clusters, but optionally in dispersed cell suspensions, confluent cell cultures, seeded on a matrix or other cell support, mixtures of the foregoing, etc.
  • the invention relates to insulin-producing cell compositions in which at least about 50% of the cells are positive for insulin production, optionally at least 75% of the cells are positive for insulin production and preferably at least 85%, 90% or 95% of the cells are positive for insulin production, hi certain embodiments, most of the cells, and preferably greater than 80%>, 90% or 95% of the cells, that produce insulin are negative for other pancreatic hormones that are not naturally produced by native pancreatic insulin producing cells, such as glucagon.
  • an insulin-producing cell composition comprises at least about 1000 nanograms (ng) of insulin per milligram of total protein, optionally at least about 5000 nanograms of insulin per milligram of total protein and preferably at least about 10000 nanograms of insulin per milligram of total protein, h embodiments where the insulin- producing cell composition comprises islet-like cell clusters of roughly 300-400 ⁇ m in diameter, the clusters produce greater than 0.2 ng of insulin per hour, and preferably greater than 0.5 ng of insulin per hour.
  • insulin production by the insulin-producing cell composition is stimulated by exposure to glucose.
  • insulin-producing cell compositions comprise cells that are positive for one or more of the following markers: insulin (any of the various chains), islet- 1, nestin, neurogenin 3, PDX1, GLUT2 and glucokinase.
  • insulin any of the various chains
  • islet- 1 nestin
  • neurogenin 3 PDX1, GLUT2
  • glucokinase glucokinase.
  • at least about 50% of the cells in an insulin-producing cell composition are not proliferative.
  • Proliferating cells may be detected by a variety of ways known in the art, including staining with Ki67, a nuclear marker of proliferating cells, or incorporation of labeled nucleotide (e.g. tritiated thymidine or bromodeoxyuridine).
  • insulin-producing cell compositions do not form neoplastic growths when implanted in a subject.
  • the insulin- producing cell compositions of the invention produce a neoplastic growth in a fewer than 30% of implanted subject, optionally in fewer than 1% of implanted subjects and preferably in fewer than 0.1% of implanted subjects.
  • the invention relates to methods for ameliorating, in a subject, a condition related to insufficient pancreatic function by administering to the subject an effective amount of an insulin producing cell composition.
  • a sufficient amount of insulin-producing cell composition is administered to a subject to cause an increase in blood insulin levels or an improvement in glucose homeostasis.
  • Glucose homeostasis may be tested by administering a dose of glucose and monitoring the kinetics with which blood glucose levels decline.
  • Conditions related to insufficient pancreatic function include the various forms of diabetes mellitus (e.g.
  • an insulin- producing cell composition may not produce a permanent ameliorating effect, and periodic dosing, such as on a weekly, monthly or yearly basis may be beneficial.
  • an effective dose of insulin-producing cell composition comprises administering at least about one islet-like cell cluster of the invention (or an equivalent number of cells) per islet that is naturally present in the subject organism.
  • mice have about 300-500 islets
  • rats have about 3000-5000 islets
  • humans have about 1,000,000 islets
  • a preferred dosage is about 300-500 islet-like cell clusters for a mouse, about 3000-5000 islet-like cell clusters for a rat and about 1,000,000 isletlike cell clusters for a human.
  • the number of islets per organism is proportional to average body mass (20-30 grams, mouse, 200-300 grams, rat, 60-70 kilograms, human) and it may be desirable to administer a dosage that is proportional to body mass of the subject, hi instances when an islet-like cell cluster is less efficient at producing insulin than a native islet, or where an insulin-producing cell composition is subject to cell mortality (e.g. in the case of host immune system-mediated rejection), the dosage may be increased proportionally.
  • the invention relates to therapeutic compositions comprising insulin-producing cell compositions and methods for making such therapeutic compositions, as well as the use of insulin-producing cell compositions in the manufacture of a medicament for the treatment of subjects having insufficient pancreatic function.
  • Therapeutic compositions may include an insulin-producing cell compostions disclosed herein and/or made by the methods disclosed herein, as well as mixtures comprising such insulin-producing cell compositions and a therapeutic excipient.
  • therapeutic excipients include matrices, scaffolds or other substrates to which cells may attach (optionally formed as solid or hollow beads, tubes, or membranes), as well as reagents that are useful in facilitating administration (e.g. buffers and salts), preserving the cells (e.g. chelators such as sorbates, EDTA, EGTA, or quaternary amines or other antibiotics), or promoting engraftment.
  • Cells may be encapsulated in a membrane to avoid immune rejection. By manipulation of the membrane permeability, so as to allow free diffusion of glucose and insulin back and forth through the membrane, yet block passage of antibodies and lymphocytes, normoglycemia may be maintained (Sullivan et al. (1991) Science 252:718).
  • hollow fibers containing cells may be immobilized in a polysaccharide alginate. (Lacey et al. (1991) Science 254:1782). Cells may be placed in microcapsules composed of alginate or polyacrylates. (Lim et al. (1980) Science 210:908; O'Shea et al. (1984) Biochim. Biochys. Ada.
  • the site of implantation of insulin-producing cell compositions may be selected by one of skill in the art. hi general, such as site preferably has adequate blood perfusion to allow the cells to sense blood conditions and secrete hormones and other factors into the general circulation. Exemplary implantation sites include the liver (via portal vein injection), the peritoneal cavity, the kidney capsule and the pancreas.
  • the invention relates to methods employing insulin-producing cell compositions disclosed herein.
  • methods of the invention relate to the identification of pancreatic developmental markers. For example, expression patterns of established markers of endoderm and islet development may be monitored at one or more stages of differentiation into insulin-producing cell compositions. Markers may be assessed using standard methods, including Northern blotting, RT-PCR, in situ hybridization (ISH), immunohistochemistry (LHC) as well as nucleic acid or protein array or microarray-based methods. In certain embodiments, monitoring production of one or more gene products will be useful to identify candidate cell- surface proteins for FACS-based purification strategies for insulin-producing cell precursors.
  • ISH in situ hybridization
  • LHC immunohistochemistry
  • Yet another aspect of the present invention provides methods for screening various compounds for their ability to modulate insulin-producing cells, such as, for example, by affecting growth, proliferation, maturation or differentiation, or by affecting insulin production, secretion or storage, as well as compounds that may improve graft performance (e.g. result in a longer-lasting graft, improved insulin production, or changes in proteins that interact with the host immune system).
  • the subject cells can be used to screen various compounds or natural products, such as small molecules or growth factors. Such compounds may be tested for essentially any effect, with exemplary effects being cell proliferation or differentiation, insulin production, or cell death.
  • insulin-producing cells may be used to test the activity of compounds/factors to promote survival and maturation, and further, since certain cells produced according to methods disclosed herein have one or more properties of islet cells, specifically ⁇ -cells, such cells may be used to identify factors ( or g enes) that regulate production, processing, storage, secretion, and degradation of insulin or other relevant proteins (like LAPP, glucagon, including pro-glucagon, GLPs, etc) produced in pancreatic islets.
  • factors or g enes
  • an msulin-producing cell may be modified, such as by genetic modification, to become hyperproliferative.
  • hyperproliferative cells may be contacted with compounds to identify, for example, anti- proliferative and anti-neoplastic agents (e.g. agents that inhibit cell growth or promote cell death).
  • anti- proliferative and anti-neoplastic agents e.g. agents that inhibit cell growth or promote cell death.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the compound.
  • a control assay can also be performed to provide a baseline for comparison.
  • Identification of the progenitor cell population(s) amplified in response to a given test agent can be carried out according to such phenotyping as described above. Assays such as those described above may be carried out in vitro (e.g. with cells in culture) or in vivo (e.g. with cell implanted in a subject).
  • the invention provides non-human animals that comprise an insulin-producing cell composition as disclosed herein. Such animals may be useful, for example, for screening compounds that may affect graft performance in vivo. 6. Methods for Identifying Stem Cells
  • the invention relates to methods for identifying a cell that has the potential to develop into a pancreatic cell, and particularly an insulin-producing cell.
  • the method comprises providing a stem cell line, or other multipotent cell line, and differentiating the cell line so as to obtain an insulin-producing cell composition.
  • the differentiating cells are mixed with a cell of interest.
  • the differentiation of the cell of interest may then be assessed.
  • a cell of interest that is able to differentiate into an insulin-producing cell is a cell that has the potential to develop into an insulin-producing cell.
  • the cell may be assessed for the production of other pancreatic products, such as glucagons, to identify cells that have the potential to develop into other types of pancreatic cells.
  • a pancreatic tissue e.g. ductal tissue, adult pancreatic tissue, fetal pancreatic tissue, etc.
  • clumps of cells or single cells are used as the cell of interest in the above method embodiments, thereby permitting a rapid screen of pancreatic cells for candidate pancreatic progenitors.
  • insulin-producing cell compositions and methods for generating such compositions may be used to assess the developmental potential of a cell of interest.
  • the developmental potential of a cell of interest may be determined by mixing t he c ell o f i nterest w ith c ells d uring t he process o f m aking a n i nsulin-producing cell composition.
  • the cell of interest is then tracked (for example by a transgenic marker) to determine the types of cells that arise from it.
  • the cell of interest is mixed with differentiating ES cells at one of the culturing steps preceding treatment with a PI3K inhibitor.
  • culture systems for making insulin-producing cell compositions may be used as part of an assay to identify candidate pancreatic endocrine precursor cells.
  • pancreatic progenitor cells could comprise detecting isolated single cells, h certain embodiments, cell compositions in the process of differentiating into insulin-producing cell compositions provide the appropriate cellular microenvironment to permit pancreas-derived endoderm to integrate and differentiate.
  • cells of a pancreatic tissue are fractionated and mixed, either as populations of cells or as single cells, into cells being differentiated into insulin-producing cell compositions. Cells of the pancreatic tissue that develop into msulin-producing cells are candidate pancreatic stem cells.
  • Example 1 Rescue of diabetes phenotypes with insulin-producing grafts derived from ES cells
  • This example describes experimental strategies for developing insulin-producing cell clusters (ICCs) from ES cells that increase circulating insulin levels, improve glucose homeostasis, and rescue survival in an experimental model of diabetes mellitus.
  • ICCs insulin-producing cell clusters
  • the stem cells were the JM1 mouse ES cell line described in M. Qiu et al., Genes Dev 9,
  • the undifferentiated mouse ES cells (stage 1) were cultured on a feeder layer of irradiated mouse embryonic fibroblasts with media containing Knockout-DMEM, penicillin-streptomycin, 0.0007%) ⁇ -mercaptoethanol, 2 mM L -glutamine, 100 mM non-essential amino acids (Gibco, Rockville, MD), 15% FCS (Hyclone, Logan, UT), and 1000 U/ml Leukemia Inhibitory Factor (LIF; Chemicon, Temecula, CA). Media was changed daily for 4 days, and then cells were harvested and placed in fresh culture plates (Fisher, Santa Clara, CA).
  • stage 2 After 2 days cells were placed in low-adhesion plates (Corning International, Corning, NY) and cultured in media without LIF (stage 2). Resultant embryoid bodies were transferred to culture dishes and allowed to adhere, then cultured for 6 days (stage 3) in ITSFn serum-free media (S. H. Lee et al., Nature Biotechnol. 18, 675, 2000). Cells were transferred to plates coated with poly-L-ornithine (Sigma, Saint Louis, MO) and fibronectin (Gibco) and cultured for 6 days (stage 4) in N 2 media (S. H. Lee et al., Nature Biotechnol.
  • ICCs were cultured i n N 2 m edia s upplemented w ith B27 a nd 1 0 m M n icotinamide ( Sigma).
  • S tage 5 NL ICCs were cultured in N 2 media supplemented with 10 mM nicotinamide and 10 ⁇ M LY294002 (Calbiochem, La Jolla, CA). Media was changed during stage 5 every other day for at least 6 days. Islets were isolated by intra-ductal collagenase perfusion.
  • Applicants performed immunohistochemistry on 6 ⁇ m tissue sections prepared by microtomy (Leica, San Jose, CA) using standard protocols. Applicants used primary antibodies at the following dilutions: guinea pig anti-insulin 1:200 (Linco Research Inc., St. Charles, MO), mouse anti- glucagon 1:500 (Sigma, St.
  • RNA was prepared using an RNeasy Kit (Qiagen) and RQ1 RNase-free DNase
  • oligo dT primers (Invitrogen) were used to prime reverse transcription reactions and synthesis was carried out by Thermoscript RT (Invitrogen). PCR was performed using Taq polymerase (Applied Biosystems), and an Opti-Prime Optimization kit (Stratagene). In addition to ⁇ -actin, GAPDH expression (not shown) was used to normalize input template cDNA to analyze relative gene expression. Primer sequences for insulin, glucagon and ⁇ -actin were as described in N. Lumelsky et al., Science 292, 1389, 2001. All animal studies were performed in accordance with Stanford University Animal Care and Use guidelines.
  • mice were engrafted with 300 handpicked ICCs or received a sham transplant of saline solution in the left subcapsular renal space.
  • Stage 5NL ICC diameter was approximately 300-400 ⁇ m, 2-3 times larger than pancreatic islets, and transplantation of more ICCs in a single renal graft site was not feasible.
  • Grafts were removed after 3 weeks by unilateral nephrectomy for analysis. Survival distributions were determined using the standard product limit method described in E. L. Kaplan, P. Meier, J. Am. Stat. Assoc. 53, 457 (1958). By 3 weeks, applicants detected tumors in 4 of 4 mice transplanted with handpicked s tage 5 NB ICCs.
  • glucose tolerance testing was performed with 1 g glucose/kg body weight as described by S. K. Kim et al, Nat Genet 30, 430 (2002).
  • Undifferentiated ES cells were exposed to a sequence of conditions described above that culminated in treatment with nicotinamide and LY294002 (stage 5NL). Other cells were treated with nicotinamide and B27 supplement (stage 5NB). Direct comparison revealed that intracellular insulin content (3.7 ⁇ 1.3 ng/ICC) in stage 5NL ICCs was 7% of levels in isolated pancreatic islets (52 ⁇ 6.3 ng/ islet). Normalized to protein content, the insulin level in stage 5NL ICCs was 4900 ⁇ 820 ng/mg protein, a 3000% increase compared to the insulin level i ICCs produced by others after treatment of ES cells with nicotinamide and B27.
  • stage 5NL, but not stage 5NB, ICCs were stained by ditbizone, a chromogen (R. M. Jindal, Pancreas 11, 316, 1995) used to detect stored intracellular insulin in pancreatic ⁇ - cells (data not shown).
  • Applicants obtained similar results with a combination of wortmannin, a different PI 3K inhibitor and nicotinamide (See Example 2).
  • stage 5NL ICCs were smaller and had more contacts between insulin cells than stage 5NB ICCs (Fig.l).
  • hi stage 5NL insulin 4" cells and pancreatic ⁇ cells co-expression of glucagon, and other markers like ⁇ - fetoprotein, a marker of primitive endoderm and liver, or microtubule-associated protein 2 (MAP2) and ⁇ -tubulinlll, neuronal-specific markers, was rare or not detected (Fig. 1A-B, D-E, G-H, J-K).
  • MAP2 microtubule-associated protein 2
  • Insulin "1" cells in stage 5NL ICCs expressed numerous pancreatic ⁇ cell markers, including C-peptide, proinsulin, the transcription factor PDX1, type 2 glucose transporter (GLUT2), and glucokinase (Fig. 2A-J).
  • Reverse transcriptase-polymerase chain reaction (RT- PCR) analysis showed that isolated ICCs expressed Islet- 1, nestin, and neurogenin-3, markers also expressed in isolated pancreatic islets (Fig. 2K).
  • Immunohistochemical detection of Ki67 a nuclear marker of proliferating cells, demonstrated that the majority of cells in stage 5NL ICCs were not proliferating, similar to cells in pancreatic islets (Fig. IM, N).
  • Our analyses Fig.
  • stage 5NL cells lacking insulin that produced carboxypeptidase A (carbA), a pancreatic exocrine gene product not detected in ICCs described in p revious s tudies.
  • carboxypeptidase A carboxypeptidase A
  • stage 5NB ICCs were comprised mainly of MAP2 or ⁇ -tubulin III expressing cells (Fig. II, L).
  • Ki67 staining revealed that approximately 25%> of cells in stage 5NB ICCs were proliferating (Fig. 1O).
  • insulin* cells produced from our methods displayed hallmark molecular features of pancreatic ⁇ cells and comprised ICCs with remarkable similarities to pancreatic islets.
  • 5NL ICCs released substantially more insulin than the 5NB ICCs (Fig. 4)
  • ES cell-derived ICCs To determine the feasibility of transplanting ES cell-derived ICCs to treat diabetes mellitus, applicants performed a series of ICC grafting experiments in immunocompromised recipient mice. Undifferentiated or partially-differentiated ES cell grafts often form tumors, particularly teratomas. In contrast, transplantation of pancreatic islets does not produce these lethal tumors.. Stage 5NB and stage 5NL ICC fates were monitored following engraftment in the left subcapsular renal space of NOD scid mice. Within 3 weeks after transplantation of stage 5NB ICCs, tumors formed (Fig. IP) with features characteristic of teratomas (data not shown).
  • stage 5NL grafts maintained high levels of insulin and C-peptide expression in clustered cells that did not co-express glucagon, and did not form tumors (Fig. 1Q, R).
  • h ⁇ sulin + cells from renal grafts appeared larger than those in stage 5NL ICCs prior to engraftment (Fig IB, R).
  • Exposure to PI 3K inhibitors can reduce cell size, suggesting that engraftment of stage 5NL ICCs in an environment lacking LY294002 permitted cell enlargement.
  • ICCs were transplanted into NOD scid mice with streptozotocin (STZ)-induced diabetes mellitus. 7 days after STZ injection, NOD scid mice were engrafted with ICCs or received a sham transplant. In mice with STZ-induced diabetes, the most striking benefit of engraftment with 300 stage 5NL ICCs was complete rescue of survival (Fig. 3A). Endogenous pancreatic insulin content in ICC-transplanted mice remained severely reduced at the completion of these transplantation experiments (Table 1), indicating that recovery o f host pancreatic insulin secretion did not account for the observed phenotypic rescue.
  • STZ streptozotocin
  • ICC-engrafted mice also achieved improved glucose regulation compared to sham-transplanted control mice in glucose tolerance tests after overnight fasting (Fig. 3C).
  • Our data support the conclusion that transplantation of stage 5NL ICCs in this diabetes model increased levels of circulating insulin, leading to better glucose regulation and maintenance of body mass. Rapid restoration of normoglycemia in mice with STZ-induced diabetes by pancreatic islet transplantation requires engraftment of 500-1000 islets per mouse; thus, further improvements in glycemic control by ICC transplantation are feasible, either by increasing ICC graft mass, or insulin expression.
  • Cell culture medium should be prepared using aseptic technique.
  • Solutions should be combined in a Nalgene Filter Unit with a 0.2 ⁇ m filter and need not be mixed before applying a vacuum. Once prepared the solutions should be stored in the refrigerator at least 4°C on the top shelf for up to a week.
  • the tissue culture are kept at 37°C and 5% CO .
  • Fibronectin [preferred supplier Gibco-BRL #33010-018, alternate Sigma #F-4759]
  • LIF Leukemia Inhibitory Factor
  • Non-Essential Amino Acids NEAA lOOx [Gibco-BRL #11140-050]
  • Penicillin-Streptomycin (Pen/Strep) lOOx [Gibco-BRL #15140-122]
  • PBS Phosphate-Buffered Saline
  • Stage 0 MEF media (Mouse Embryonic Feeder)
  • ITSFn media Insulin, Transferrin, sodium Selenite, Fibronectin
  • Stage 1 Day 3 (4) 1. Change media of ES cells.
  • Stage 2 Day 1 (6) 1. Pre-warm DisA to room temperature.
  • Pre-warm KO LTF(-) media and transfer all the cells from their individual wells into a 50 ml tube using a 10ml pipet. Let the cells settle for 5-10 minutes. Aspirate off media.
  • Stage 2 Day 3 (8) (early evening) 1. Collect embryoid bodies and wash each well with 3 ml of media.
  • Stage 3 Day 3 (12) 1. Warm ITSFn media. 2. Aspirate off the old ITSFn media
  • N 2 media (-B27, bFGF, and nicotinamide). 2. Prepare an aliquot of N 2 media with 50X B27 and 100X bFGF.
  • Stage 4 Day 3 (18) 1. Stage 4 media change
  • Stage 5 Day 5 (26) 1. Stage 5 media change.
  • Example 3 Withdrawal of PI3K Inhibitor Induces ICC Growth mES cells were processed through the 5NL stage as described above. A portion of the
  • ICCs were then transferred to a 6NI medium, lacking the PI3K inhibitor.
  • the effects of removal of the inhibitor are shown in Fig 5, as well as Fig. IR.
  • PI3K inhibition followed by PI3K activation induces ICC growth.
  • Immunofluorescence staining of ICCs treated with only stage 5NL conditions (A) or stage 5NL and stage 6NI conditions (B) express insulin (green).
  • Greyscale conversion of A and B, C and D respectively demonstrates not only an overall increase in insulin expression (black) but also an overall increase in cell growth with the addition of stage 6 conditions.
  • These insulin expressing cells at stage 6NI also expressed C-peptide, but as in stage 5NL, there was no significant expression of MAP2, ⁇ -tubulinm, glucagon, or ⁇ -fetoprotein in stage 6 NI ICCs.
  • Example 4 Influence of different manipulations on ICC growth and insulin production The effects of different combinations of PI3K inhibitors and their withdrawal on insulin production by ICCs was tested.
  • Table 2 Comparison of insulin and protein content following treatment of ES cell-derived ICCs with different combinations of growth factors.
  • ROSA-derived cells were mixed with suspended stage 3 or stage 4 ICC cells and co-cultured until stage 5 when cell clusters were visible, ⁇ -galactosidase+ c ells i n ICCs w ere d etected w ith X gal ( Fig 6 ).
  • Cell culture medium should be prepared using aseptic technique.
  • Solutions should be pre-warmed in the 37 °C water bath unless otherwise noted. Solutions should be combined in a Nalgene Filter Unit with a 0.2 ⁇ m filter and need not be mixed before applying a vacuum.
  • the solutions should be stored in the refrigerator at 4 °C on the top shelf for up to a week.
  • tissue culture Incubators, Refrigerators, and Freezers
  • the tissue culture are kept at 37 °C and 5% CO .
  • Fibronectin [preferred supplier Gibco-BRL #33010-018, alternate Sigma #F-4759]
  • LIF Leukemia Inhibitory Factor
  • Non-Essential Ammo Acids NEAA lOOx [Gibco-BRL #11140-050]
  • Penicillin-Streptomycin (Pen/Strep) lOOx [Gibco-BRL #15140-122]
  • PBS Phosphate-Buffered Saline
  • Stage 0 MEF media (Mouse Embryonic Feeder) 88 ml DMEM (+ Glutamax and Na Pyruvate)
  • Stage 1 and 2 KO media, LIF (+ or -)
  • ITSFn media Insulin, Transferrin, sodium Selenite, Fibronectin
  • Stage 2 Day 2 (7) 1. Pre-warm KO LIF(-) media and transfer all the cells from their individual wells into a 50 ml tube using a 10ml pipet. Let the cells settle for 5-10 minutes. Aspirate off media.
  • DMEM/F-12 media Pre-warm DMEM/F-12 media. 3. Very gently aspirate offthe solution from the attached embryoid bodies. Very gently add 3 ml of DMEM F-12 and aspirate to wash the cells and eliminate any FCS. 4. Add 3 ml of ITSFn media to each dish and incubate at 37 °C.
  • N 2 media (- B27, bFGF, and nicotinamide).

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Abstract

L'invention concerne en partie des compositions cellulaires produisant de l'insuline, des procédés pour produire des compositions cellulaires produisant de l'insuline, au moyen d'un inhibiteur de prolifération de cellules, et des procédés thérapeutiques et non thérapeutiques destinés à être utilisés dans des compositions cellulaires produisant de l'insuline.
PCT/US2003/023346 2002-07-25 2003-07-25 Compositions cellulaires produisant de l'insuline et procedes associes WO2004010933A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013034436A1 (fr) 2011-09-08 2013-03-14 Hexal Ag Composition pharmaceutique à administration orale contenant une statine
US10494608B2 (en) 2015-04-24 2019-12-03 University Of Copenhagen Isolation of bona fide pancreatic progenitor cells
US11060062B2 (en) 2017-10-26 2021-07-13 University Of Copenhagen Generation of glucose-responsive beta cells

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6165733A (en) * 1996-05-23 2000-12-26 Chiron Corporation γ II adaptin
US6242666B1 (en) * 1998-12-16 2001-06-05 The Scripps Research Institute Animal model for identifying a common stem/progenitor to liver cells and pancreatic cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6165733A (en) * 1996-05-23 2000-12-26 Chiron Corporation γ II adaptin
US6242666B1 (en) * 1998-12-16 2001-06-05 The Scripps Research Institute Animal model for identifying a common stem/progenitor to liver cells and pancreatic cells

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013034436A1 (fr) 2011-09-08 2013-03-14 Hexal Ag Composition pharmaceutique à administration orale contenant une statine
US10494608B2 (en) 2015-04-24 2019-12-03 University Of Copenhagen Isolation of bona fide pancreatic progenitor cells
US11613736B2 (en) 2015-04-24 2023-03-28 University Of Copenhagen Isolation of bona fide pancreatic progenitor cells
US11060062B2 (en) 2017-10-26 2021-07-13 University Of Copenhagen Generation of glucose-responsive beta cells

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